Chromatography column support

ABSTRACT

Herein is reported the use of a chromatography column support comprising at least one plane of symmetry, one axis of symmetry, at least three legs, at least three straight connectors, whereby the connectors define a plane that is perpendicular to the axis of symmetry of the support, whereby the connectors are connected to each other at the axis of symmetry, whereby each leg is connected to a connector, whereby each leg is perpendicular to the plane defined by the connectors, whereby all legs are on the same side of the plane defined by the connectors for stabilizing the packing of a chromatography column.

Herein is reported a chromatography column support which allows amovable frit to be positioned within a chromatography column at adesired position. The support comprises a multitude of legs and amultitude of connectors connected to the upper end of the legs providinga support over the entire diameter of the column for a movable frit.

BACKGROUND OF THE INVENTION

The design of large-scale preparative chromatographic processes can bevery complex and can involve many factors that are not primarily“chromatographic”. These factors include method of column packing,hydrodynamic pressure drop, and flow distribution. These ultimatelyfactor into the economics of the process.

Stickel, J. J. and Fotopoulos, A. (Biotechnol. Prog. 17 (2001) 755-751)report pressure-flow relationships for packed beds of compressiblechromatography media at laboratory and production scale.

For gel filtration columns at technical scale the problem of compactionof the gel filling resulting in a reduced capacity can be avoided by thearrangement of separation walls inside the chromatography column with adirection parallel to the wall of the column as reported in EP 0 005708.

In EP 0 176 891 a column for high pressure chromatography is reportedcomprising additional support elements for the stabilization of thecolumn packing.

In U.S. Pat. No. 3,398,512 a chromatographic column is reported. Thechromatography apparatus as reported therein comprises a column of thetype having a gas inlet, a gas outlet, at least one column wallinterconnecting the gas inlet and gas outlet and confining solidparticulate packing material, and means for preventing and reducingcomponent profile distortion by straightening or reshaping componentprofiles after they have sustained distortion.

In U.S. Pat. No. 5,770,061 it is outlined that by providingchromatography columns with plates which are permeable to the liquidflow but carry the filling material, the filling material can be dividedinto several superimposed layers, thus diminishing the pressuredifference over the total height of the filling material (see e.g. GB1,203,439, U.S. Pat. No. 3,539,505). Attempts have also been made toeliminate the disadvantages of large columns by means of various wallsdisposed in the column and having the direction of the vertical axis ofthe column. These walls divide the column into several parallel sectionsof a smaller cross-section, said sections extending continuouslythroughout the filling bed or throughout the almost entire bed (see e.g.EP 0 005 708, U.S. Pat. No. 5,124,133, U.S. Pat. No. 3,298,527).

In WO 91/014490 a slurry compressor is reported that facilitatesobtaining a homogeneous absorbent bed packing in liquid chromatographycolumns, thus improving the efficiency of the columns and increasing theaccuracy of the results obtained thereby.

In U.S. Pat. No. 6,440,301 an apparatus which facilitates the use ofstandard sized and configured laboratory glassware as chromatographycolumn is reported. It is also reported a stainless matrix holder, theholder defining a central opening and further defining acircumferential, reduced diameter grooved engaged by a gasket such as anO-ring.

In U.S. Pat. No. 3,374,606 is reported a method and apparatus forchromatographic separations comprising sieve plates that may be arrangedin any sequence or position within the packed bed or separatory materialin a chromatography column, thereby separating the column into a seriesof separatory zones.

It has been reported in U.S. Pat. No. 5,770,061 that the capacity andseparation result of large separation columns can be markedly improvedby arranging in the column an inner structure which is light,self-supporting and easy to install in the column. In more detail it isreported a separation column which is characterized in that its fillingmaterial space comprises in the vertical direction at least two zonesbetween which no means that would hinder vertical flow exist and whichare divided by means of substantially vertical walls into separatesections, the zones being so dimensioned that the upper edge of thewalls in the uppermost zone is below the upper surface of the columnfilling material in the operating conditions of the column.

In U.S. Pat. No. 4,351,621 a connector for combination furniture isreported. A high velocity chromatography column flow distributor isreported in EP 1 606 615. In JP 2008-241 624 a chromatography column-useliquid introducing unit is reported. A chromatography device is reportedin JP 62-042051.

SUMMARY OF THE INVENTION

One aspect as reported herein is a chromatography column supportcomprising

-   -   at least one plane of symmetry,    -   a single axis of symmetry,    -   at least three legs,    -   at least three straight connectors,

-   whereby the connectors form a plane that is perpendicular to the    axis of symmetry of the support,

-   whereby the connectors are connected to each other at the axis of    symmetry or at a circle around the axis of symmetry,

-   whereby each leg is connected to a connector,

-   whereby each leg is perpendicular to the plane formed by the    connectors,

-   whereby all legs are on the same side of the plane formed by the    connectors.

In one embodiment the legs are plates comprising a multitude of holes.In a further embodiment the support comprises three legs and oneconnector, whereby the connector is Y-shaped. In also an embodiment thesupport comprises at least four legs and at most sixteen legs andcomprises four connectors.

In one embodiment at least one of the connectors and one of the legs arecombined in one unit. In one embodiment each of the connectors iscombined with a leg in a unit.

In one embodiment the chromatography column support comprises more legsthan connectors.

In one embodiment the chromatography column support comprises

-   -   at least three legs that each form a unit with a connector,    -   at least three legs that do not form a unit with a connector,    -   at least one leg that is circular around the axis of symmetry.

In one embodiment the chromatography column support is a single entity.

In one embodiment the chromatography column support comprises multipleentities.

In one embodiment the connectors have a width of less than 8.0 mm.

In one embodiment the chromatography column support has a diameter ofless than 12.5 cm.

In one embodiment the chromatography column support is made of stainlesssteel, or silicone, or polytetrafluoroethylene, or glass.

Another aspect as reported herein is a device comprising

-   -   a chromatography column,    -   at least one movable frit, and    -   at least one support as reported herein,

-   wherein the moveable fit and the chromatography column support are    inside the chromatography column, and

-   whereby the moveable fit is above the chromatography column support,    when the chromatography column is placed vertically and viewed from    the side.

Another aspect as reported herein is the use of a chromatography columnsupport as reported herein for the stabilization of a chromatographycolumn packing from inside the packing.

DETAILED DESCRIPTION OF THE INVENTION

Herein is reported a chromatography column support comprising

-   -   one axis of symmetry,    -   at least one plane of symmetry that is perpendicular to the axis        of symmetry,    -   at least three legs that are perpendicular to the plane of        symmetry,    -   at least three straight connectors,

-   whereby the connectors define the plane of symmetry that is    perpendicular to the axis of symmetry of the support,

-   whereby the connectors are connected to each other at or around the    axis of symmetry,

-   whereby each leg is connected to a connector,

-   whereby each leg is perpendicular to the plane defined by the    connectors,

-   whereby all legs are on the same side of the plane defined by the    connectors,

-   whereby the plane defined by the connectors is not solid.

It has been found that the contact interface between the moveable fitplaced upon the chromatography column support and the chromatographycolumn, in which the device is placed, shall not interfere with theproperties of the supported moveable frit.

It has been found that for the construction of the chromatography columnsupport it has to be accounted for the fact that it is placed within thechromatography material and, thus, it is surrounded by it. Thus, thechromatography column support is constructed in a way to minimize thedisturbance of the chromatography material during the column packingprocess. Additionally it is constructed in a way to minimize thedisturbance of the mobile phase flowing though the chromatographymaterial during a chromatographic separation process.

It has been found that the chromatography column support as reportedherein can be made as a single piece (e.g. by casting the moldedmaterial in a casting mold) or from multiple pieces (e.g. by connectingthe pieces together, such as screwing or bonding).

The chromatography column support as reported herein is not affixed tothe chromatography column. It can be removed from the column without theneed to first detach it from the column.

The chromatography column support as reported herein supports thechromatography column packing inside the packing. In contrast thereto,chromatography columns as well as top and/or bottom plates inside thechromatography column support the chromatography column packing from theoutside. The chromatography column support as reported herein forms aframework inside the chromatography column packing. This frameworksupports the stability and homogeneity of the chromatography materialpacking from inside the packing. In contrast a common chromatographycolumn supports the chromatography material packing only from theoutside.

Thus, a chromatography column packing comprising a chromatography columnsupport as reported herein has improved stability compared to achromatography column packing in a conventional chromatography columnwithout the chromatography column support as reported herein.

The term “one connector and one leg are combined in one unit” denotesthat the presence of an additional connector is obsolete as the leg hasthe same length as the connector and is connected to otherlegs/connectors at or around the axis of symmetry.

In a liquid column chromatography purification method the chromatographymaterial is located within a column housing (chromatography column) anddenoted as “stationary phase”. To enable a stationary phase to interactwith substances/polypeptides in a solution applied to it, the stationaryphase is surrounded by/embedded in a “mobile phase”. The term “mobilephase” denotes a liquid, e.g. a buffered, aqueous solution, a mixture ofwater and an organic solvent, or an organic solvent, which is used inthe chromatographic purification method in which a stationary phase isemployed.

Different chromatography methods are well established and widespreadused for polypeptide recovery and purification, such as affinitychromatography with microbial proteins (e.g. protein A or protein Gaffinity chromatography), ion exchange chromatography (e.g. cationexchange (carboxymethyl resins), anion exchange (amino ethyl resins) andmixed-mode exchange), thiophilic adsorption (e.g. withbeta-mercaptoethanol and other SH ligands), hydrophobic interaction oraromatic adsorption chromatography (e.g. with phenyl-sepharose,aza-arenophilic resins, or m-aminophenylboronic acid), metal chelateaffinity chromatography (e.g. with Ni(II)- and Cu(II)-affinitymaterial), size exclusion chromatography, and electrophoretical methods(such as gel electrophoresis, capillary electrophoresis) (see e.g.Vijayalakshmi, M. A., Appl. Biochem. Biotech. 75 (1998) 93-102).

In a column chromatography separation or purification of a crudepolypeptide normally a chromatography column comprising a chromatographymaterial and a mobile phase is employed. The mobile phase is forcedthrough the chromatography column and therewith through thechromatography material by applying pressure to the mobile phase.Mediated by the mobile phase the pressure is also applied to thechromatography material whereby a pressure drop from the inlet of thechromatography column to the outlet of the chromatography column isestablished. At the outlet of the chromatography column the pressure hasdropped to the outside atmospheric pressure. Thus, to the upper fractionof the chromatography material in the chromatography column the highestpressure force is applied.

The applied pressure normally depends on the one hand on the particlesize of the chromatography material as well as on the viscosity of themobile phase as a constant flow through the chromatography column is setbut not a constant pressure. Generally the pressure increases withdecreasing chromatography material particle size. At a constant flowrate through the chromatography material a change in viscosity of themobile phase, e.g. during the regeneration or cleaning of thechromatography material, results in a change of the pressure applied tothe chromatography material. The chromatography material in general isnot a pressure insensitive material, i.e. it can be compressed andexpands after a compression. Therefore, with an increase of the appliedpressure the chromatography material is compressed and the height of thechromatography material inside the chromatography column, i.e. the bedheight, is reduced. Likewise with a decrease of the applied pressure thechromatography material expands again and the height of thechromatography material inside the chromatography column increases atmost to the height before the application of the pressure. Thiscompression and expansion of the chromatography material is at the sametime a macroscopic process of the entire chromatography material and amicroscopic process of the individual particles of the chromatographymaterial. With increasing numbers of such compression-expansion-cyclesthe particles of the chromatography material break down into smallerparticles. With decreasing particle size of the particles of thechromatography material the chromatography material packing gets morecompact and, therewith, at the same time the pressure required formaintaining a constant liquid phase flow through the chromatographycolumn, i.e. the chromatography material, increases. This in turn againresults in a further break down of the chromatography material particlesresulting again in an increased pressure and so on.

A chromatography column separation generally can be operated up to amaximum pressure. When this upper pressure limit is reached thechromatography column packing has to be replaced in its entirety.

The chromatography column support as reported herein provides for anumber of advantages.

The support as reported herein is movable within the column, i.e. it canbe placed anywhere along the axis of the column. Thus, the height of thesupport and therewith the height of the corresponding horizontal sectionof the chromatography column is variable and can be adjusted to theproperties of the chromatography material used with the support.Additionally, as the support is movable within the column damage to theinner wall of the chromatography column is prevented.

The support as reported herein can be made of any material that can beused in column chromatography, such as Teflon (polytetrafluoroethylene),stainless steel, silicone, or glass. In one embodiment thechromatography column support as reported herein is made ofpolytetrafluoroethylene or stainless steel or silicone or glass. Thisallows for the provision of an inert support that does not interfere orinteract with the chromatography material (column packing) as well asthe substances to be separated. In one embodiment the chromatographycolumn support is made of a chromatographically inert material.

Because the support as reported herein is not affixed to thechromatography column the support can be applied to and also removedfrom the column in an easy way. Additionally because the support isremovable from the column it can be easily and efficiently cleaned andsanitized. In one embodiment the chromatography column support in thedevice as reported herein is freely moveable within the chromatographycolumn. In one embodiment the chromatography column support in thedevice as reported herein is within the chromatography column but notaffixed to the inner wall of the chromatography column.

Further, with the support as reported herein it is possible to provide aflexible, generally applicable addition to existing chromatographycolumns. Therewith the costs required to expand the applicability ofexisting chromatography columns are reduced.

The support as reported herein can be provided in many differentvariations in diameter and height and is therewith applicable to amultitude of different chromatography columns. But the diameter of thechromatography column support as reported herein is always less than theinner diameter of the chromatography column in order to ensure a freemovability inside the column either during the packing of the columnand/or the use of the column.

If the legs of the support are in the form of a plate an additionalstabilization effect of the chromatographic material can be provided. Inthis embodiment the legs and the connectors form a single unit and,thus, the chromatography column support comprises at least three legsthat are connected to each other at or around the axis of symmetry.

Generally the support as reported herein can be used in combination witha movable frit. In this combination the support can provide astabilizing effect throughout the entire chromatography material insidethe column.

The support as provided herein improves the packing process as ahomogeneous packing of the chromatography material can be effectedalready prior to the application of the support. After the use of thecolumn (single-use column) or when the chromatography material has to bereplaced (multi-use column) the support can easily be removed with thechromatography material, just as with a column not comprising a supportas reported herein. It is also possible to make the packing process withthe support as reported herein already introduced into the respectivehorizontal section of the chromatography column.

It has been found that the support as reported herein can be used withany chromatography column giving the chromatography material therein aninner structure and support without the need to modify thechromatography column.

Thus, one aspect as reported herein is the use of a chromatographycolumn support as reported herein for supporting chromatography materialin a chromatography column.

In one embodiment is the support made of stainless steel, or silicone,or polytetrafluoroethylene, or glass.

In one embodiment the support comprises

-   -   at least one plane of symmetry,    -   one axis of symmetry,    -   at least three legs,    -   at least three straight connectors,

-   whereby the connectors define a plane that is perpendicular to the    axis of symmetry of the support,

-   whereby the connectors are connected to each other at or around the    axis of symmetry,

-   whereby each leg is connected to a connector,

-   whereby each leg is perpendicular to the plane defined by the    connectors,

-   whereby all legs are on the same side of the plane defined by the    connectors.

In one embodiment the legs are plates and that the connectors and thelegs form a unit in which the upper edge of the respective leg is theconnector.

In one embodiment the legs are perforated plates.

In one embodiment the support comprises one or more circular legs thathave different radii with respect to each other and with respect to theaxis of symmetry.

In one embodiment a lateral flow between the sections defined by thelegs of the support is possible.

Advantageous is further the unique symmetric structure of the support,especially when made of an inert material.

By the open architecture of the support, i.e. due to the absence of apermanent fixation to the inner chromatography column wall thechromatography material is provided with an inner structure withoutintroducing compartments that are tightly closed to other compartmentsinside the column. By the open structure (i) the packing of thechromatography material can be obtained in a more homogeneous form, (ii)the formation of tension inside the packing can be prevented, (iii) flowand mass transport is ensured over the entire cross-section of thechromatography column, (iv) an easy packing and de-packing process ispossible. Thus, one aspect as reported herein is the use of achromatography column support as reported herein for providing an innerstructure to a chromatography material in a chromatography column.

Furthermore the supported packing region can be from the bottom of thecolumn up to the top without any non-supported areas in between or aboveor below the chromatography material packing.

Additionally each of the horizontal sections provided by a combinationof a support as reported herein and a movable frit can be packedindividually enabling the packing of a chromatography column from thebottom to the top. In addition in each horizontal section a differentchromatography material can be introduced, thus, providing a means for aso called hybrid-chromatography.

In combination with a movable frit the support as reported hereinprovides for a vertical as well as horizontal supportation of thechromatography material inside the chromatography column. Thus, oneaspect as reported herein is the use of a chromatography column supportas reported herein for providing vertical and horizontal support of thechromatography material inside a chromatography column.

Thus, the support as reported herein provides a means for using thecurrently used chromatography columns in a more flexible way and at thesame time minimizing the costs associated with flexibility as only thenovel support has to be added.

In the upper part of FIG. 1 one embodiment of the support as reportedherein which has four legs in the form of planes (full areas) is shown.In this embodiment the legs and the connectors are combined in a singleunit and the support does not comprise additional connectors. The upperedge of the leg corresponds to the connector. The planes of the legs arein a 90° angle to each other and to the plane defined by the upper edgesof the legs (i.e. the connectors). The diameter of the support and theouter area of the legs (i.e. the outer edge of the legs which is theedge in the direction of the column and perpendicular to the planedefined by the upper edges/connectors) can be adapted to any columndiameter, so that the support can be introduced and removed into anycolumn without friction and damaging the inner surface of the column.The height of the support as reported herein is variable and can beadjusted to the specific application. The bottom area of the support canbe adapted to the base on which the support is to be placed. The base isa frit in most cases. The base supports the support and carries hisweight without the column wall being involved. The upper area of thechromatography column support is formed in a way that either it issuitable as a base for a moveable column fit (moveably chromatographycolumn separator) or the upper adapter of the column can be put thereon.It is therefore ensured that the complete packing is stabilized by thesupport as reported herein. Thus, one aspect as reported herein is theuse of a support as reported herein to stabilize the packing of achromatography column. If a moveable column separator is used, anothersupport, optionally in a different form, can be put on the separator atmost until the maximum column length reached. However, it also possiblethat only a part of the chromatography column packing is equipped withthe support as reported herein.

In the lower part of FIG. 1 one embodiment of the support as reportedherein is shown. This support comprises six legs each in form of a planewhich are divided up symmetrically. The upper edge of each of the sixlegs represents the six connectors with which the legs form a unit inthis embodiment. The legs are attached to each other at a circle aroundthe axis of symmetry of the support. The pressure expected inside acolumn and the function of the support which is demanded determines thenumber of legs. These are dependent on the used chromatography mediumand the process conditions.

In the upper part of FIG. 2 one embodiment of the support as reportedherein is shown. In this embodiment the legs are in the form of a planeand form a unit together with the connectors which are represented bythe upper edges of each of the legs. Characteristic for this embodimentare the longish openings in the planes of the legs of the support. Thisform offers an improved support for the chromatographic material and isespecially suited for soft gels. Thus, one aspect as reported herein isthe use of a chromatography column support as reported in thisembodiment for stabilizing the packing of soft chromatography gels.Without being bound by theory it is assumed that the chromatographicmaterial can find more support/contact area in the narrow rooms. Theload and the pressure are absorbed by the support and passed on to thelower layers of the column packing in reduced form. Thus, one aspect asreported herein is the use of a chromatography column support asreported herein for adsorbing pressure inside a chromatography columnpacking. Another characteristic of this embodiment is the crosswaysnetworking which is obtained. A lateral flow between the individualsections made up by the legs of the support is therefore possible. Thus,in one embodiment of the support as reported herein a lateral flowbetween the individual sections defined by the legs of the support andthe axis of symmetry of the support is possible. This should provide animproved mass transfer and column performance. The homogeneity of thepacket during the column life also remains unchanged. The appearingtensions spread out better in column bed, which is only partlyinterrupted.

In the middle part of FIG. 2 one specific embodiment of the support asreported herein is shown. In this embodiment the legs are in the form ofa perforated plate. Also in this embodiment each of the connectors formsa single unit with the individual legs. In this embodiment some of thelegs are connected to each other at the axis of symmetry of the support.In this embodiment the support comprises additional legs that are notconnected to each other around or at the axis of symmetry. This supportis characterized by the fact that holes inside the planes of the legs ofthe support provide the crossways networking. In this embodiment thelegs are made of perforated plates. In this embodiment the supportfurther comprises at least two, especially three, circular legs (andround connectors) that all have different radii with respect to the axisof symmetry of the support. Thus, in one embodiment a first number oflegs (the connectors) is connected to each other at the axis of symmetryof the support and all legs (connectors) are connected to each other bythe circular legs (round connectors) of the support. In one embodimentthe support comprises three circular legs (round connectors), wherebythe first circular leg (round connector) has a radius with respect tothe axis of symmetry of the support of one sixth of the total diameterof the support, the second circular leg (round connector) has a radiusof one third of the total diameter of the support, and the thirdcircular leg (round connector) has a radius of half the total diameterof the support. All legs are also connected to each other with thefurther round connectors. The relationship of the free areas and thewall spaces in this embodiment is different from that in the beforeoutlined embodiments. In this case more sections result for the packinghere, what can be beneficial for large column diameters. Thus, oneaspect as reported herein is the use of a connector comprising circularlegs and perforated plates as legs for stabilizing the column packing oflarge diameter chromatography columns. The term “large diameterchromatography column” denotes a chromatography column with an innerdiameter of 15 cm or more.

In the lower part of FIG. 2 one exemplary embodiment of the support asreported herein is shown. Herein the support is shown inside achromatographic column. The column comprises a support as reportedherein and a moveably chromatography column fit placed thereon. Theseelements are flexible and not permanently connected to the column wallor other column components or to each other. In this embodiment thesupport comprises three legs in form of a rod and three connectorsconnected to each other at the axis of symmetry of the support.

In FIG. 3 an exemplarily chromatographic column which is equipped withseveral different supports as reported herein is shown. The column inaddition comprises a number of moveable chromatography columnseparators. The column wall offers only the guidance for the supportsand the moveable column separators which glide along the chromatographycolumn wall while they are moved. The column wall does not have to bemodified. None of the elements is permanently connected or affixed tothe column wall. Such a column can also be packed with differentchromatographic materials in the individual horizontal sections made upof a pair of support and separator. Thus, one aspect as reported hereinis the use of a support as reported herein and a moveable frit to definehorizontal sections in a chromatography column. The composition of allthe used elements can be changed very simple and every time. Unpackingthe column can be carried out in a conventional way. The used supportsand moveable chromatography column separators can simply be removed fromthe column together with the column packing. All used equipment partscan independently of each other be cleaned or partly replaced.

In FIG. 4 a pressure flow diagram of a chromatography column comprisinga DEAE-Sepharose chromatographic material is shown. The left curve showsthe typical behavior of a DEAE-Sepharose chromatographic material incolumns which are packed in a conventional way. In the right curve thebehavior of a column equipped with two supports as reported herein andtwo chromatographic column separators is shown. It can be seen that themaximum flow rate of the conventionally packed column is less than halfof the flow rate which has been achieved with the column comprising asupport as reported herein (24 l/h instead of 55 l/h). It can be seenthat a triple decompression takes place (from 1.5 bar to 0.5 bar) in theflow area of 22 l/h which represents a typical process flow (details ofthe column without a support as reported herein: 4 l slurry of DEAESepharose, bed height: 24 cm; used eluent: 0.0005 mol (conductivity 5mS) KCl solution; details of the column comprising a support as reportedherein: height of the used support: 8 cm, strength of the usedseparator: 5 mm, 4 l slurry of DEAE Sepharose distributed on threecompartments, bed height: 24 cm, used eluent: 0.0005 mol (conductivity 5mS) KCl solution).

In FIG. 5 a pressure flow diagram of a chromatography column comprisingan HA-Ultrogel chromatographic material is shown. The left curve showsthe typical behavior of HA-Ultrogel in columns which are operatedwithout a support as reported herein. In the right curve the behavior ofa column comprising two supports as reported herein and two moveablechromatographic column separators is shown. It can be seen that themaximum flow of the column without the support as reported herein isless than the flow rate of the column comprising a support as reportedherein (8 l/h instead of 15 l/h) (details of the column without asupport as reported herein: 4 l slurry of HA-Ultrogel, bed height: 24cm; used eluent: 0.0005 mol (conductivity 5 mS) KCl solution; details ofthe column comprising a support as reported herein: height of the usedsupport: 8 cm, strength of the used separator: 5 mm, 4 l slurry of DEAESepharose distributed on three compartments, bed height: 24 cm, usedeluent: 0.0005 mol (conductivity 5 mS) KCl solution).

Thus, another aspect as reported herein is a device comprising

-   -   a chromatography column,    -   at least one movable chromatography column separator, and    -   at least one support as reported herein.

In one embodiment the device comprises two chromatography columnseparators and two supports as reported herein. In another embodimentthe device comprises three chromatography column separators and threesupports as reported herein. In another embodiment the separator and thesupport both have an outer diameter that is less than the inner diameterof the chromatography column. In also an embodiment the diameter of thesupport and the separator is of from 90% to 99% of the inner diameter ofthe chromatography column. All the embodiments of the support asreported herein and outlined above are also embodiments of the supportscomprised in the device as reported herein. Specific embodiments of theseparators comprised in the device as reported herein are outlinedbelow.

The presence of one chromatography column separator divides thechromatography column in an upper chromatography column chamber and alower chromatography column chamber. The separator has a variableposition within the chromatography column. This “movability” is providedfor by a guide ring. Thus, the separator can slide vertically within thecolumn and the separator is embedded in the chromatography material.

A chromatography column separator consisting of a guide ring and a fitmounted therein does not interfere with the chromatographical separationprocess. The separator allows e.g. that only a fraction of thechromatography material has to be exchanged when the maximum operatingpressure is reached, without the need to replace the entirechromatography column packing. That is, the chromatography columnseparator permits that the chromatography material in an upperchromatography column chamber can be exchanged without interfering withthe chromatography material in a lower chromatography column chamber.The partial removal of the chromatography material is possible as theseparator on the one hand divides the total chromatography material inthe chromatography column in distinct fractions and on the other handprevents the packed chromatography material in the lower chromatographycolumn chamber from being perturbed upon the removal of thechromatography material in the upper chromatography column chamber.Thus, at least the fraction of the chromatography material that is notexposed to the maximum pressure changes and, thus, is not torn up can beused further without a negative impact on the separation efficiency.But, by retaining a fraction of the chromatography material a cost ofgoods reduction can be achieved.

The chromatography column separator comprises a guide ring into which afit made of any inert material can be mounted. An “inert material” is amaterial that does not interfere with the chromatography separationprocess, i.e. a chromatogram obtained with a chromatography columncontaining one or more chromatography column separators as reportedherein is identical to a chromatogram obtained with a chromatographycolumn containing no chromatography column separators under/withotherwise identical conditions. Such inert materials are e.g. metal,especially stainless steel, silicone, polypropylene, polyethylene,polytetrafluoroethylene, sintered materials or combinations thereof,especially polytetrafluoroethylene coated stainless steel.

In FIG. 6 exemplary chromatography column separators are shown. In FIG.6 a) a separator with a single frit is depicted comprising a frit (1)and a fitting (2). In FIG. 6 b) a separator with an upper frit (3) and alower frit (4) and a fitting (2) is shown. In FIG. 6 c) the verticalcross-section of the guide ring of the separator comprising two axiallysymmetric cross-section areas (5 and 6) is shown, wherein each of theaxially symmetric cross-section areas has a) a tapering structure,wherein the tapering is from the outside to the inside of the guidering, and b) a notch (8) with an opening directed to the inside of theguide ring for mounting a frit.

In one embodiment the chromatography column separator comprises a guidering and a fit mounted into the guide ring.

In one embodiment the guide ring is of circular shape and for use in aliquid chromatography column. In another embodiment the guide ring has avertical cross-section comprising two axially symmetric cross-sectionareas (5 and 6), wherein each of the axially symmetric cross-sectionareas has

-   -   a) a tapering structure, wherein the tapering is from the        outside to the inside of the guide ring, and    -   b) a notch (8) with an opening directed to the inside of the        guide ring for mounting a frit.

In one embodiment the notch is a rectangular notch. In a furtherembodiment each of the cross-section areas has a triangular shape andthe longest side (7) has a length of at least 1.5 times the diameter ofthe notch (8). In a further embodiment the ring is made of rubber,plastic, silicone, polytetrafluoroethylene, polyethylene, orpolypropylene.

In one embodiment the fit is

-   -   a) a single frit, or    -   b) two frits with an upper frit and a lower frit.

In another embodiment a) the frit has a pore size of from 1 μm to 20 μm,or b) each of the frits has a pore size of from 1 μm to 20 μmindependently of each other whereby the pore size of the upper frit issmaller than the pore size of the lower frit. In another embodiment thefrit is made of metal, silicone, polypropylene, polyethylene,polytetrafluoroethylene, sintered materials or combinations thereof. Ina further embodiment the separator comprises distance holders allattached to one side of the separator.

In one embodiment the chromatography column separator is characterizedin that

-   -   a) the separator separates a chromatography column section in an        upper chromatography column chamber and a lower chromatography        column chamber, and    -   b) the separator has a variable position within the        chromatography column by sliding along the inner wall of the        chromatography column.

In one embodiment the chromatography column separator comprises onefrit, in another embodiment the separator comprises an upper fit and alower frit. In a further embodiment the chromatography column separatoror the upper fit or the lower fit has a pore size of from 1 μm to 20 μm,whereby the pore size of the upper frit is smaller than the pore size ofthe lower frit. In another embodiment the frit is made of metal,silicone, polypropylene, polyethylene, polytetrafluoroethylene, sinteredmaterials or combinations thereof.

The fitting or guide ring has a circular shape with a cross-section thatcan have any shape as long as it has a rectangular notch for taking upthe frit. For example, in one embodiment the cross-section of thefitting has the shape of a triangle with a rectangular notch for takingup the frit in the corner of the triangle with the biggest inside angle.In a further embodiment the fitting or guide ring has the cross-sectionor provides cross-sectional areas in form of a triangle, in anotherembodiment of a rectangular triangle, wherein the frit is attached tothe corner of the triangle with an inner angle of 90°. In anotherembodiment the cross-section area of the guide ring has a trapezoid fromwith the rectangular notch for taking up the frit being at the shorterside of the parallel sides. In one embodiment the fitting has the formof a rectangle, in another embodiment of a rectangle with inner anglesof 90°, 90°, 80° and 100°. In one embodiment are the inner angles of 80°and 100° are at the upper side or at the bottom side of the rectangle.In one embodiment the longest side of the guide ring is the outer edgeof the separator and has contact to the chromatography column wall whenthe separator is placed inside a chromatography column. In anotherembodiment the longest side has a vertical orientation. In still anotherembodiment the cross-section areas of the fitting or guide ring have theform of a rectangle with inner angles of 90° with the rectangular notchfor taking up the frit in one of the shorter sides or in case of asquare in one of the sides. The side with the notch is the side of thefitting directing to the center of the separator and likewise thechromatography column or in other words the notch is in the side of thefitting or guide ring that is parallel to the flow direction of themobile phase and that has a diameter smaller than the outer diameter ofthe frit. The guide ring has besides the preventing of liquid phase andchromatography material particles passing the separator beside frit thefunction to prevent the canting and therewith stalling of the entireseparator in the chromatography column during the compression andexpansion of the chromatography material upon the applying of theoutside pressure. The separator is placed inside the chromatographymaterial packed into a chromatography column. The separator can be movedfreely and placed exactly inside the column as it can slide along theinner wall of the chromatography column. This is useful during thepacking of the chromatography column and for removing the separator fromthe chromatography column. In one embodiment the verticalcross-sectional areas of the guide ring have the form of a triangle ortrapezoid in which the guide ring has a tapering structure, wherein thetapering is from the outside to the inside of the guide ring, that isthe guide ring is at its outer edge higher than at its inner edge or atthe notch, respectively. In one embodiment the outer edge of the guidering has a height that is at least 1.5 times the height of the notch. Inanother embodiment the outer edge of the guide ring has a height that isat least 1.5 times, or two times, or three times, or more than threetimes the height of the notch.

If the separator comprises an upper frit and a lower frit in oneembodiment the fitting or guide ring is a single fitting or ring and inanother embodiment the fitting or guide ring is made of an upper fittingor ring and a lower fitting or ring. In the latter case the two fittingsor rings have in one embodiment a contact-area comprising the lower sideof the upper fitting or ring and the upper side of the lower fitting orring, whereby the contact sides are flat, i.e. have no notch or groove,and are in line with the lower side of the upper fit and the upper sideof the lower frit, i.e. the lower side of the upper fitting and thelower side of the upper frit form a single surface without offset andlikewise the upper side of the lower fitting or ring and the upper sideof the lower frit form a single surface without offset whereby bothsurfaces are parallel.

Generally the smallest inner diameter of the fitting or guide ring or ofthe upper fitting and of the lower fitting is smaller than the outerdiameter of the frit, i.e. the fitting or guide ring extends over theouter perimeter of the fit towards the center of the chromatographycolumn.

The packing of a chromatography column with a chromatography materialwith a support and/or an embedded chromatography column separator can besplit up into two packing phases. The packing can be started with thepacking of a first fraction of the chromatography material into thecolumn according to general procedures. Afterwards the support and thechromatography column separator can be placed in and on top of the firstfraction of the chromatography material. Finally the second fraction ofthe chromatography material can be packed into the column on top of theseparator according to general procedures. This packing method is apacking from the bottom to the top. In contrast columns not containing asupport and a separator are packed from the top requiring among otherthings higher packing pressure. Thus, the chromatography column supportas reported herein provides a means for packing a chromatography columnin two sequential steps if one support and one separator are used, or inthree or more sequential steps if two or more supports and separatorsare used. With the support and the separator the column can be dividedin an upper chamber and a lower chamber (one support and one separator)or a lower chamber, a middle chamber, and an upper chamber (two supportsand two separators) whereof each itself is equivalent to achromatography column with reduced chromatography material bed height.With the dividing of the chromatography column in smaller chambers thevolume (of the chromatography material in one chamber) to surface (ofthe chamber) ratio is changed, i.e. lowered, and the stability of thechromatography material packing is increased.

The following examples and figures are provided to aid the understandingof the present invention, the true scope of which is set forth in theappended claims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

Aspects and embodiments as reported herein

One aspect as reported herein is a chromatography column supportcomprising

-   -   at least one plane of symmetry,    -   one axis of symmetry,    -   at least three legs,    -   at least three straight connectors,

-   whereby the connectors define a plane that is perpendicular to the    axis of symmetry of the support,

-   whereby the connectors are connected to each other at the axis of    symmetry or at a circle around the axis of symmetry,

-   whereby each leg is connected to a connector,

-   whereby each leg is perpendicular to the plane defined by the    connectors,

-   whereby all legs are on the same side of the plane defined by the    connectors.

One aspect as reported herein is a chromatography column supportcomprising

-   -   one axis of symmetry,    -   at least one plane of symmetry that is perpendicular to the axis        of symmetry,    -   at least three legs that are perpendicular to the plane of        symmetry,    -   at least three straight connectors,

-   whereby the connectors define a plane that is perpendicular to the    axis of symmetry of the support,

-   whereby the connectors are connected to each other at or around the    axis of symmetry,

-   whereby each leg is connected to a connector,

-   whereby each leg is perpendicular to the plane defined by the    connectors,

-   whereby all legs are on the same side of the plane defined by the    connectors,

-   whereby the plane defined by the connectors is not solid.

In one embodiment the connectors are plates.

In one embodiment the legs are plates and the connectors are the upperedge of the respective plate.

In one embodiment at least one of the connectors and one of the legsform a single unit, whereby the legs are plates and the connector is theupper edge of the plate. I.e. the connector is defined by the upper edgeof the plate.

In one embodiment each of the connectors forms a single unit with anindividual leg.

In one embodiment each of the connectors forms a single unit with a leg.In this embodiment each of the legs forms a single unit with a connectorand, thus, the chromatography column support comprises units that areconnected to each other at or around the axis of symmetry.

In one embodiment some of the legs are connected to each other at theaxis of symmetry of the support.

In one embodiment the plates are perforated plates.

In one embodiment the support comprises additional legs that are notconnected to each other at or around the axis of symmetry.

In one embodiment the support comprises one or more circular legs havingdifferent radii with respect to the axis of symmetry of the support.

In one embodiment a lateral flow between the sections defined by thelegs of the support and the axis of symmetry of the support is possible.

In one embodiment the support comprises three legs and one connector,whereby the connector is Y-shaped and the legs are in form of rods.

In one embodiment the support consists of three legs in form of rods anda Y-shaped connector.

In one embodiment the support consists of three legs in form of platesthat are connected to each other at or around the axis of symmetry.

In one embodiment the support consists of four legs in form of rods andan X-shaped connector.

In one embodiment the support consists of four legs in form of platesthat are connected to each other at or around the axis of symmetry.

In one embodiment the support consists of six legs in form of rods andan X-shaped connector.

In one embodiment the support consists of six legs in form of platesthat are connected to each other at or around the axis of symmetry.

In one embodiment the support consists of

-   -   i) four first legs in form of plates that are connected to each        other at the axis of symmetry,    -   ii) four second legs in form of plates that are not connected to        each other and to the four first legs at the axis of symmetry,        and    -   iii) three circular legs that are connected to each of the first        and second four legs but not to each other and that have        different radii with respect to each other and the axis of        symmetry.

In one embodiment the plates are perforated plates.

In one embodiment the support comprises at least four legs and at mostsixteen legs and comprises four connectors.

In one embodiment the chromatography column support comprises more legsthan connectors.

In one embodiment the chromatography column support comprises

-   -   at least three legs that each form a unit with a connector,    -   at least three legs that do not form a unit with a connector,    -   at least one leg that is a circular leg around the axis of        symmetry.

In one embodiment the chromatography column support is a single entity.

In one embodiment the chromatography column support comprises multipleentities.

In one embodiment the connectors have a width of less than 8.0 mm.

In one embodiment the chromatography column support has a diameter ofless than 12.5 cm.

In one embodiment the chromatography column support is made of achromatographically inert material.

In one embodiment the chromatography column support is made of stainlesssteel, or silicone, or polytetrafluoroethylene, or glass.

One aspect as reported herein is a device comprising

-   -   a chromatography column,    -   at least one movable frit, and    -   at least one support as reported herein,

-   wherein the moveable fit and the chromatography column support are    inside the chromatography column, and

-   whereby the moveable fit is above the chromatography column support,    when the chromatography column is placed vertically and viewed from    the side.

In one embodiment the chromatography column support is freely moveablewithin the chromatography column.

In one embodiment the chromatography column support in the device asreported herein is within the chromatography column but not affixed tothe inner wall of the chromatography column.

One aspect as reported herein is the use of a chromatography columnsupport as reported herein for the stabilization of a chromatographycolumn packing from inside the packing.

One aspect as reported herein is the use of a chromatography columnsupport as reported herein for supporting chromatography material insidea chromatography column.

One aspect as reported herein is the use of a chromatography columnsupport as reported herein for providing an inner structure to achromatography material in a chromatography column.

One aspect as reported herein is the use of a chromatography columnsupport as reported herein for providing vertical and horizontal supportof the chromatography material inside a chromatography column.

One aspect as reported herein is the use of a support as reported hereinto stabilize the packing of a chromatography column.

One aspect as reported herein is the use of a chromatography columnsupport comprising perforated plates as combined legs and connectors orcomprising plates as legs which comprise longish openings forstabilizing the packing of soft chromatography gels.

One aspect as reported herein is the use of a chromatography columnsupport for adsorbing pressure inside a chromatography column packing.

One aspect as reported herein is the use of a connector comprisingcircular legs and perforated plates as legs for stabilizing the columnpacking of large diameter chromatography columns.

One aspect as reported herein is the use of a support as reported hereinand a moveable frit to define horizontal sections in a chromatographycolumn.

DESCRIPTION OF THE FIGURES

FIG. 1 Exemplary embodiments of the support as reported herein: upper:element which has four legs in the form of full areas, these in 90°angles stand to each; lower: element which has six legs.

FIG. 2 Exemplary embodiments of the support as reported herein: upper:element with four legs comprising longish openings in the leg areas;middle: element with round holes in the legs providing crosswaysnetworking; lower: element which has three legs inside a chromatographycolumn

FIG. 3 Scheme of a chromatographic column comprising several differentsupports as reported herein and several chromatography columnseparators.

FIG. 4 Flow diagram of a DEAE-Sepharose chromatography column: leftcurve: column without a support as reported herein; right curve: columnwith a support as reported herein.

FIG. 5 Flow diagram of a HA-Ultrogel chromatography column: left curve:column without a support as reported herein; right curve: column with asupport as reported herein.

FIG. 6 Exemplary chromatography column separators: a) separator with asingle frit comprising a frit (1) and a fitting (2); b) separator withan upper frit (1) and a lower frit (3) and an upper fitting (2) and alower fitting (4); c) vertical cross-section of the guide ring of theseparator comprising two axially symmetric cross-section areas (5 and 6)each having i) a tapering structure, wherein the tapering is from theoutside to the inside of the guide ring, and i) a notch (8) with anopening directed to the inside of the guide ring for mounting a frit.

FIG. 7 Pressure values during the regenerations and cycle numbers of achromatography column not comprising the support as reported herein.

FIG. 8 Pressure values during the regenerations and cycle numbers of achromatography column comprising the support as reported herein.

EXAMPLES Example 1 Packing of a Chromatography Column Comprising aSupport as Reported Herein

During the packing process of columns which are equipped with theflexible bed support elements as reported herein the homogeneousinserting and distribution of the chromatography medium is veryimportant. There are two possibilities to reach a homogeneous packing.The first is to put the flexible bed support element in the column firstand inserting the slurry afterwards. In this case the slurry musthomogeneously be brought in into the column. The other possibility isbringing in the slurry into the column first, homogenizing it in thecolumn and afterwards introducing the bed support element by putting itdirectly into the slurry in the column. During the settling ahomogeneous column bed forms and encloses the element. After thespecified settling time can either an intermediate column fit be put onthe bed support element or the column can be locked with an adapter. Ifan intermediate column frit is used, another flexible bed supportelement can be put on top and the packing of the next column compartmentcan be started. The process can be repeated until the complete columnheight is filled/packed.

Example 2 Chromatographic Separation Using a Conventional ChromatographyColumn not Comprising a Support as Reported Herein—Cycle Number andPressure Characteristics for the use of the Chromatography Material

Herein the life cycle of a conventionally packed HPLC column is shown.It can be seen from FIG. 7 that the pressure directly after packing isabout 40 bars. This increases after the fortieth regeneration cycle toabout 70 bars.

Packing details:

-   -   Column diameter: 30 cm    -   Column type: dynamic compression without bed support elements        and intermediate column frits    -   Bed height: about 40 cm    -   Used chromatographic material: Vydac C4    -   Flow rate: 162 l/h

Pressure values during the regenerations and cycle numbers are shown inFIG. 7.

Example 3 Chromatographic Separation Using a Conventional ChromatographyColumn Comprising a Support as Reported Herein—Cycle Number and PressureCharacteristics for the use of the Chromatography Material

Herein is shown a HPLC column with a support as reported herein and anintermediate column frit. It can be seen that a lower initial pressureafter packing compared to the column of Example 2 can be obtained.Altogether a more stable pressure course can be obtained over the usecycles. Also a maximum cycle number of 98 can be reached without theneed to repack the column. The intermediate column frit without thesupport as reported herein fulfills a bed support function at the HPLCmedia which are compressible only in a very low measure.

Packing details:

-   -   Column diameter: 15 cm    -   Column type: dynamic compression with intermediate column frit    -   Bed height: about 40 cm    -   Used chromatographic material: Vydac C4    -   Flow rate: 35 l/h

Pressure values during the regenerations and cycle numbers are shown inFIG. 8.

1.-5. (canceled)
 6. A chromatography column support comprising: at leastone plane of symmetry, one axis of symmetry, at least three legs, atleast three straight connectors, wherein the connectors define a planethat is perpendicular to the axis of symmetry of the support, whereinthe connectors are connected to each other at the axis of symmetry,wherein each leg is connected to a connector, wherein each leg isperpendicular to the plane defined by the connectors, and wherein alllegs are on the same side of the plane defined by the connectors.
 7. Thesupport according to claim 6, wherein the legs are plates and that theconnectors and the legs form a unit in which the upper edge of therespective leg is the connector.
 8. The support according to claim 6,wherein the legs are perforated plates.
 9. The support according toclaim 6, wherein the support comprises one or more circular legs thathave different radii with respect to each other and with respect to theaxis of symmetry.
 10. The support according to claim 6, wherein alateral flow between the sections defined by the legs of the support ispossible.
 11. The support according to claim 6 comprising three legs andone connector, whereby the connector is Y-shaped and the legs are rods.12. The support according to claim 6, comprising at least four legs andat most sixteen legs and comprising four connectors.
 13. The supportaccording to claim 6, wherein support comprises more legs thanconnectors.
 14. The support according to claim 6, wherein the supportcomprises i) four first legs in form of plates that are connected toeach other at the axis of symmetry, ii) four second legs in form ofplates that are not connected to each other and to the four first legsat the axis of symmetry, and iii) three circular legs that are connectedto each of the first and second four legs but not to each other and thathave different radii with respect to each other and the axis ofsymmetry.
 15. A device comprising a chromatography column, at least onemovable frit, and at least one support according to claim 6, wherein themoveable fit and the chromatography column support are inside thechromatography column, and wherein the moveable fit is above thechromatography column support, when the chromatography column is placedvertically and viewed from the side.
 16. A method of stabilizing achromatography column packing comprising providing to a chromatographycolumn having packing therein a chromatography column support accordingto claim
 6. 17. A method for performing liquid chromatographypurification comprising: providing the device of claim 15; passing amobile phase having substances to be separated therein through thechromatography column by applying pressure to the mobile phase such thatthe substance are separated and eluted separately from thechromatography column.
 18. The support according to claim 7, wherein thelegs are perforated plates.
 19. The support according to claim 7,wherein the support comprises one or more circular legs that havedifferent radii with respect to each other and with respect to the axisof symmetry.
 20. The support according to claim 7, wherein a lateralflow between the sections defined by the legs of the support ispossible.
 21. The support according to claim 7 comprising three legs andone connector, whereby the connector is Y-shaped and the legs are rods.22. The support according to claim 7, comprising at least four legs andat most sixteen legs and comprising four connectors.
 23. The supportaccording to claim 8, wherein the support comprises one or more circularlegs that have different radii with respect to each other and withrespect to the axis of symmetry.
 24. The support according to claim 8,wherein a lateral flow between the sections defined by the legs of thesupport is possible.
 25. The support according to claim 8 comprisingthree legs and one connector, whereby the connector is Y-shaped and thelegs are rods.